Since the initial funding of this grant proposal, the hypocretin/orexin system has been recognized to be a hypothalamic neuropeptide system with widespread excitatory activity. Defects in this system, either presynaptically or postsynaptically, result in the sleep disorder narcolepsy in both animal models and in humans. Based on these and other observations, hypocretin (Hcrt) is now viewed as a central neurotransmitter system in the maintenance of wakefulness. During the next funding period, we propose to examine specific inputs to and outputs from the Hcrt neurons at both the cellular and behavioral levels. We hypothesize that there is an interaction between the Hcrt and the corticotropin releasing factor (CRF) systems. To further understand this relationship, we will (1) determine the origin of CRF-containing afferent innervation of the Hcrt neurons using anterograde tracing from potential CRF afferent sources combined with Hcrt immunohistochemistry and retrograde tracing from the perifornical hypothalamus with CRF-immunohistochemistry; (2) identify the mechanism of action of the excitatory effect of CRF on Hcrt cells; and (3) determine whether an intact Hcrt system is necessary for the anxiogenic effects of CRF. We will identify whether CRF is anxiogenic in hypocretin-ataxin cell knockout mice and evaluate the effect of hypocretin peptides in four anxiety models in wildtype mice. These studies will determine whether the Hcrt system is "downstream" from CRF. We will also determine the response of Hcrt neurons to stimulation by other neurotransmitters that may provide modulatory input to the Hcrt system. We will examine the mechanisms underlying GABAergic inhibition of the Hcrt neurons and determine whether endogenous inhibitory tone exists in this system. We will also examine other potential sources of modulatory input to the Hcrt cells, such as that from the hypothalamic ghrelin and galanin systems. Lastly, we will determine whether subpopulations of Hcrt neurons can be differentiated based on their efferent projections to sleep-related monoaminergic and cholinergic nuclei. To address this question, we will use dual retrograde tracing from different sleep-related regions to determine the extent of co-localization of retrograde markers within Hcrt neurons and the distribution of these markers within the Hcrt neuronal population. The information obtained will elucidate the neural networks related to the Hcrt system and thereby further our understanding of the neurobiological bases of sleep, wakefulness, and the sleep disorder narcolepsy.